smallpox dna vaccine protects nonhuman primates against lethal monkeypox

JOURNAL OF VIROLOGY, May 2004, p. 4433–4443 Vol. 78, No. 90022-538X/04/$08.00�0 DOI: 10.1128/JVI.78.9.4433–4443.2004

Smallpox DNA Vaccine Protects Nonhuman Primates againstLethal Monkeypox

J. W. Hooper,1* E. Thompson,1 C. Wilhelmsen,2 M. Zimmerman,3 M. Ait Ichou,1S. E. Steffen,1 C. S. Schmaljohn,1 A. L. Schmaljohn,1 and P. B. Jahrling4

Virology Division,1 Pathology Division,2 Veterinary Medicine Division,3 and Headquarters,4 United StatesArmy Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland 21702

Received 15 October 2003/Accepted 9 January 2004

Two decades after a worldwide vaccination campaign was used to successfully eradicate naturally occurringsmallpox, the threat of bioterrorism has led to renewed vaccination programs. In addition, sporadic outbreaksof human monkeypox in Africa and a recent outbreak of human monkeypox in the U.S. have made it clear thatnaturally occurring zoonotic orthopoxvirus diseases remain a public health concern. Much of the threat posedby orthopoxviruses could be eliminated by vaccination; however, because the smallpox vaccine is a liveorthopoxvirus vaccine (vaccinia virus) administered to the skin, the vaccine itself can pose a serious healthrisk. Here, we demonstrate that rhesus macaques vaccinated with a DNA vaccine consisting of four vacciniavirus genes (L1R, A27L, A33R, and B5R) were protected from severe disease after an otherwise lethal challengewith monkeypox virus. Animals vaccinated with a single gene (L1R) which encodes a target of neutralizingantibodies developed severe disease but survived. This is the first demonstration that a subunit vaccineapproach to smallpox-monkeypox immunization is feasible.

Due to concerns about the possible use of smallpox as abiological weapon, programs to vaccinate 500,000 military per-sonnel (mandatory) and a similar number of health care work-ers (voluntary) were implemented in December 2002. Thesmallpox vaccine used in these programs—calf lymph-derivedlive vaccinia virus (VACV) administered by scarification with abifurcated needle—is essentially the same vaccine first used �2centuries ago (27). A comparable smallpox vaccine consistingof clonal VACV grown in cell culture is being tested in clinicaltrials (29). Although VACV is highly immunogenic and isknown to confer long-lasting protective immunity to smallpox(12), the adverse events associated with the present smallpoxvaccine (i.e., Dryvax) pose a significant obstacle to successfulvaccination campaigns. Adverse events historically associatedwith VACV range from the nonserious (e.g., fever, rash, head-ache, pain, and fatigue) to life threatening (e.g., eczema vac-cinatum, encephalitis, and progressive vaccinia) (6). Seriousadverse events that are not necessarily causally associated withvaccination, including myocarditis and/or myopericarditis,have been reported during past and present smallpox vaccina-tion programs (4, 9). Several adverse cardiac events reported inthe first 4 months of the 2003 civilian and military vaccinationcampaigns prompted the Centers for Disease Control and Pre-vention to revise their recommendations for exclusion of po-tential smallpox recipients to include those persons with heartdisease or several other conditions (3). Moreover, the liveVACV vaccines are problematic because lesion-associated vi-rus at the site of vaccination is infectious and can be inadver-tently spread to other parts of the body (e.g., ocular autoinoc-ulation) and to other individuals (i.e., contact vaccinia) (6).

Although the recent smallpox vaccination programs are in-

tended to protect against bioterror events, naturally occurringpoxvirus diseases are also a growing concern because the num-ber of persons with vaccinia virus-induced immunity has beenin decline. Epidemics of human monkeypox have occurredsporadically in west and central Africa (16, 18a), and veryrecently, an outbreak of human monkeypox occurred in themidwestern United States (2). This outbreak (71 suspected and35 laboratory-confirmed cases as of 8 July 2003) may have beentransmitted from prairie dogs that were infected after beinghoused close to an imported African rodent (5). The firstmonkeypox outbreak outside Africa coupled with the severeacute respiratory syndrome (SARS) pandemic illustrates howrare zoonotic viral diseases can emerge rapidly and spread inunprotected populations.

Epidemiological studies have demonstrated that vaccinationwith VACV protects humans against smallpox and monkeypox(10a, 18a). All viruses in the genus Orthopoxvirus, family Pox-viridae, including VACV, monkeypox virus (MPOV), and va-riola virus (the virus that causes smallpox), are highly similar inthe majority of their nearly 200 proteins, which accounts su-perficially for the cross-protection among these viruses (28).However, the precise immune mechanisms by which the small-pox vaccine elicits immunity to monkeypox and smallpox re-main largely unknown. Identifying protective and problematicimmunogens will be essential for developing, testing, andbridging next-generation smallpox vaccines. In addition, iden-tifying protective immunogens might allow the development ofa subunit smallpox vaccine that affords protection with negli-gible adverse events.

Previously, we used a gene gun-delivered DNA vaccine ap-proach to test several VACV genes and gene combinations forimmunogenicity and protective efficacy in mice (13, 14). Afour-gene combination DNA vaccine (hereafter referred to as4pox DNA vaccine) protected 100% of mice challenged with alethal dose of VACV and was immunogenic in nonhuman

* Corresponding author. Mailing address: Virology Division,USAMRIID, Ft. Detrick, MD 21702. Phone: (301) 619-4101. Fax:(301) 619-2439. E-mail: [email protected].

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primates (13). There are two major forms of infectious or-thopoxvirus: the intracellular mature virion (IMV), which isinfectious when released from disrupted cells, and the extra-cellular enveloped virion (EEV), which buds from infectedcells (10a, 23). The 4pox DNA vaccine contained two IMV-specific genes (L1R and A27L) and two EEV-specific genes(A33R and B5R). The L1R and A27L immunogens are knowntargets of IMV neutralizing antibodies (25, 26, 30). Antibodiesto the B5R (but not A33R) immunogen reportedly neutralizeEEV (11, 20). The A33R immunogen is a target of antibody-dependent, complement-mediated cytolysis (A. L. Schmaljohn,unpublished observations). We hypothesize that the high levelof protection conferred when combinations of IMV and EEVimmunogens are used is due to the targeting of different formsof the virus (e.g., IMV and EEV) at different stages of infec-tion and by different mechanisms. Here, we report the resultsof a challenge experiment in which we tested the capacity ofthe 4pox DNA vaccine to protect rhesus macaques from severemonkeypox.

MATERIALS AND METHODS

Viruses and cells. The VACV Connaught vaccine strain (derived from theNew York City Board of Health strain) (21) was maintained in Vero cell (ATCCCRL-1587) monolayers grown in Eagle minimal essential medium containing 5%heat-inactivated fetal bovine serum (FBS), 1% antibiotics (100 U of penicillin/ml,100 �g of streptomycin/ml, and 50 �g of gentamicin/ml), and 10 mM HEPES(cEMEM). COS cells (ATCC CRL 1651) were used for transient-expressionexperiments. A working stock of MPOV Zaire-79 (MPOV-Z79) at 5 � 108 PFUper ml was generously provided by J. Huggins.

Gene gun vaccination. Rhesus macaques were vaccinated using the same DNAvaccine plasmids and gene gun conditions described previously (13, 14).

VACV-infected-cell lysate ELISA. Enzyme-linked immunosorbent assays(ELISAs) were performed as described previously (13).

VACV and MPOV PRNT. Plaque reduction neutralization tests (PRNT) wereperformed on Vero cells as described previously (13).

RIPA. Radioimmunoprecipitation assays (RIPAs) were performed as de-scribed previously (13).

Intravenous challenge with MPOV. Rhesus macaques were anesthetized usingtelezol at 3 to 6 mg/kg of body weight or ketamine at 10 to 20 mg/kg. MPOVsonicated for 30 s on ice and diluted in Dulbecco’s minimal essential mediumcontaining 1% FBS was injected (1 ml) into the saphenous vein using a 20- to22-gauge catheter, followed by 3 ml of saline to flush the injection site. On theindicated days, monkeys were anesthetized and weighed, and blood was drawnfrom the femoral vein using a 22-gauge needle and vacutainer tube. Pulse andoximetery were measured using a handheld digital pulse oximeter (SurgiVet,Inc., Waukesha, Wis.). Hematological values of fresh whole blood were deter-mined using a Coulter (Miami, Fla.) AcT series analyzer. Serum samples werestored at �70°C before blood chemistry values were determined using a PiccoloPoint-of-Care chemistry analyzer (Abaxis, Inc., Union City, Calif.).

Whole-blood processing for plaque assay. Approximately 300 �l of bloodcollected in K2 EDTA vacutainer tubes was transferred to microcentrifuge tubes,rapidly freeze-thawed three times, and then spun in a microcentrifuge at 10,000� g for 5 s to pellet cell debris. The supernatants were sonicated for 30 s on iceand then assayed for infectious virus by plaque assay on Vero cell monolayersstarting at a 1:100 dilution in cEMEM. The monolayers were stained 5 dayspostinfection with 1% crystal violet in 70% ethanol.

Throat swab processing for plaque assay. Sterile swabs were used to collectthroat specimens. The swabs were stored at �70°C until further use. The swabswere placed in 300 �l of medium, allowed to soak for �5 s, and then swirled toallow release of swabbed material into the medium. Throat swab suspensionswere subjected to three rapid freeze-thaw cycles and spun in a microcentrifuge at10,000 � g for 5 s to pellet cell debris. The supernatants were sonicated for 30 son ice and then assayed for infectious virus by plaque assay on Vero cell mono-layers starting at a 1:10 dilution in cEMEM. The monolayers were stained 5 dayspostinfection with 1% crystal violet in 70% ethanol.

TaqMan PCR of whole blood. DNA was extracted from frozen blood samplesby using the Aquapure DNA kit (Bio-Rad) as described previously (17). Prior

experiments had demonstrated that the material was noninfectious after a 60-min incubation at 55°C in Aquapure lysis buffer.

OPXJ7R3U (5�-TCATCTGGAGAATCCACAACA-3�) and OPXJ7R3L (5�-CATCATTGGCGGTTGATTTA-3�) and the probe OPXJ7R3P (5�-CTGTAGTGTATGAGACAGTGTCTGTGAC-3�) were selected from the variola virushemagglutinin gene (GenBank no. L22579; open reading frame J7R). The prim-ers were synthesized by using standard phosphoramidite chemistry with an ABI394 DNA-RNA synthesizer. The TaqMan probe was synthesized by PE Biosys-tems (Foster City, Calif.) and contained 6-carboxyfluorescein in the 5� end and6-carboxytetramethylrhodamine and a phosphate in the 3� end.

5� nuclease PCR assay. The 5� nuclease PCR and amplification conditionswere carried out using Platinum Quantitative PCR SuperMix-UDG (Invitrogen)as described previously (17). All reactions included at least one positive controlthat contained 5 fg (�25 copies) of MPOV genomic DNA and one no-templatecontrol. The 5-fg positive control for each run established the threshold cycle(Ct) value for positivity. Samples yielding Ct values which marginally exceededthe threshold value were retested. If the Ct value was confirmed to exceed thethreshold after retesting, the sample was considered negative (i.e., the samplecontained �25 gene copies).

Construction of Escherichia coli expression plasmids containing VACV L1R,A33R, B5R, and A27L genes. The genes coding for L1R, A33R, B5R, and A27Lfrom VACV strain Connaught were amplified from constructs described previ-ously (13, 14) using PCR and Pfx polymerase (Invitrogen) as described by themanufacturer. The primer sets used (5�-GGCATATGGGTGCCGCAGCAAGC-3� and 5�-GGCTCGAGTCAGTTTTGCATATCCG-3�, 5�-GGCATATGATGACACCAGAAAACG-3� and 5�-GGCTCGAGTTAGTTCATTGTTTTAACAC-3�, 5�-GGCATATGAAAACGATTTCCGTTGTTACG-3� and 5�-GGCTCGAGTTACGGTAGCAATTTATGG-3�, and 5�-GGCCATGGACGGAACTCTTTTCCCCG-3� and 5�-GGCTCGAGCTCATATGGACGCCGTCC-3�) werecomplementary to the 5� and 3� ends of the gene sequences (italics) of VACVL1R, A33R, B5R, and A27L, respectively, and contained the recognition sitesfor the restriction enzymes NdeI, NcoI, and XhoI (underlined). The ampli-cons obtained from the PCRs were cloned directly into pCR-Blunt II-TOPO(Invitrogen) as described by the manufacturer, and the resulting clones werescreened by restriction analysis. Plasmid DNA containing the desired VACVinserts was digested with the appropriate restriction enzymes (NdeI and XhoIfor L1R, A33R, and B5R; NcoI and XhoI for A27L). The inserts digestedwith NdeI and XhoI were subcloned into pET-16b, and the inserts digestedwith NcoI and XhoI were subcloned into pET-21b. The VACV genes in thefinal constructs, pET-L1R(VACV), pET-A33R(VACV), pET-B5R(VACV),and pET-A27L(VACV), were sequenced using a model 3100 genetic analyzer(Applied Biosystems) to ensure that no changes had occurred during sub-cloning procedures.

Expression of the vaccinia virus L1R, A33R, B5R, and A27L proteins in E. coli.The A33R, B5R, and A27L proteins were expressed in the E. coli strain BL21-CodonPlus(DE3)-RP-X (Stratagene). The L1R protein was expressed under thesame conditions as the aforementioned proteins; however, the L1R protein wasexpressed in the E. coli strain Rosetta-gami(DE3) (Novagen). Competent cellswere transformed with either pET-L1R(VACV), pET-A33R(VACV), pET-B5R(VACV), or pET-A27L(VACV) and selected for growth on Luria broth(LB)-ampicillin plates. Individual colonies were used to inoculate a 100-ml LBculture containing carbenicillin (50 �g/ml). The cells were grown to saturation at37°C and used to inoculate 3 liters of LB containing carbenicillin (50 �g/ml). Thecells were grown at 37°C to an A600 of 0.6; VACV protein expression was inducedby the addition of isopropyl-1-thio--D-galactopyranoside (final concentration, 1mM) for 3 h at 37°C. Cells were harvested by centrifugation (6,000 � g for 10min) and resuspended to a density of 0.2 g/ml in 50 mM Tris-HCl (pH 8.0)–150mM NaCl–0.1% NP-40–5 mM -mercaptoethanol. Cells expressing L1R proteinwere resuspended in 50 mM Tris-HCl (pH 8.0)–150 mM NaCl–0.1% NP-40. Thecells were rapidly frozen using liquid nitrogen and stored at �70°C.

Purification of VACV L1R, A33R, B5R, and A27L proteins. All protein prep-arations were treated identically with the following exceptions: the L1R proteinwas purified in the complete absence of -mercaptoethanol, and the A27Lprotein was purified using a 5-ml HiTrap chelating HP column (AmershamBiosciences). Frozen cell suspensions were quickly thawed in a 25°C water bath.Thawed suspensions were treated with rLysozyme (final concentration, 7.5 kU/ml) (Novagen) and rocked for 20 min at room temperature. The followingpurification steps were carried out at 4°C. Suspensions were lysed by sonicationusing a Branson model 450 sonifier with the microtip attachment and centrifugedat 30,000 � g for 1.5 h. The pellets were discarded, and the supernatants weredialyzed overnight against 2 liters of P buffer (50 mM sodium phosphate [pH7.5]–500 mM NaCl–5 mM -mercaptoethanol). The dialysates were clarified bycentrifugation at 30,000 � g for 1 h. Prior to being loaded, individual 1-ml

4434 HOOPER ET AL. J. VIROL.

HiTrap chelating HP columns were prepared according to the manufacturer’sinstructions and equilibrated with P buffer. Separate columns were loaded withlysates containing L1R, A33R, B5R, or A27L protein and washed with 5 columnvolumes of P buffer and then with 5 column volumes of P buffer plus 50 mMimidazole. The L1R, A33R, B5R, and A27L proteins were eluted with P bufferplus 500 mM imidazole to yield the final fractions. VACV protein-containingfractions were identified by Western analysis using monoclonal antibodies spe-cific for pentahistidine or VACV L1R, A33R, or A27L protein. The concentra-tions of these partially purified VACV proteins were determined by UV absor-bance at 280 nm using the extinction coefficients 1.52, 0.87, 0.82, and 8.27 A280

mg�1 ml � cm�1, which were calculated from the amino acid sequences of theL1R, A33R, B5R, and A27L proteins, respectively.

Immunogen-specific ELISA. Histidine-tagged VACV A27L, L1R, B5R, andA33R proteins were expressed in E. coli and purified from E. coli using themethods described above. Antigen diluted in 0.1 M carbonate buffer, pH 9.6, wasused to coat 96-well ELISA plates (100 �l per well). The concentrations ofpurified A27L, L1R, B5R, and A33R proteins used were 50, 250, 50, and 100ng/well, respectively. Purified histidine-tagged human Tsg101 protein or a trun-cated hantavirus nucleocapsid was used as a negative control antigen. Antigenwas adsorbed to the ELISA plates overnight at 4°C. The plates were washed onetime with PBS plus 0.05% Tween 20 (wash buffer), blocked for 1 h at 37°C withwash buffer containing 5% FBS plus 3% goat serum (blocking buffer), washedonce, and incubated for 1 h at 37°C with antibody diluted in blocking buffercontaining 20 �g of E. coli lysate/ml to reduce background. The plates werewashed three times, incubated for 1 h at 37°C with peroxidase-labeled goat-anti-monkey immunoglobulin G (Kirkegaard & Perry Laboratories, Inc., Gaithers-burg, Md.) diluted in blocking buffer, washed as before, and incubated in 100 �lof 2,2�-azino-di(3-ethylbenzthiazoline-6-sulfonate) substrate/well. After 10 to 30min at room temperature, the colorimetric reaction was stopped by the additionof 100 �l of 0.2 N phosphoric acid/well. The optical density (OD) at 405 nm wasdetermined by an ELISA plate reader. Nonspecific binding was controlled for bysubtracting OD values obtained on negative control antigen from OD valuesobtained on purified VACV antigens. End point titers were determined as thehighest dilution with an OD greater than the mean OD value from negativecontrol serum sample wells (1:50 dilution) plus 2 standard deviations.

Research was conducted in compliance with the Animal Welfare Act andother federal statutes and regulations relating to animals and experiments in-volving animals and adhered to principles stated in the Guide for the Care andUse of Laboratory Animals (23a). The facility where this research was conductedis fully accredited by the Association for Assessment and Accreditation of Lab-oratory Animal Care International.

RESULTS AND DISCUSSION

Booster DNA vaccination and challenge with MPOV. Thechallenge experiment included four groups: group 1 consistedof three monkeys vaccinated with the 4pox DNA vaccine,group 2 consisted of two monkeys vaccinated with the L1RDNA vaccine, group 3 (negative controls) consisted of threemonkeys vaccinated with a Hantaan virus DNA vaccine (13,15), and group 4 (positive controls) consisted of two monkeysvaccinated with the human smallpox vaccine (Dryvax). TheL1R DNA vaccine was tested to determine the degree to whichvaccination with a single immunogen eliciting IMV-neutraliz-ing antibodies could confer protection. The DNA vaccineswere administered by gene gun. The identification numbers,sexes, weights, and vaccination histories of the monkeys areshown along with summarized challenge result data in Table 1.Five weeks before challenge, all monkeys except the monkeysvaccinated with Dryvax and one of the negative controls(CH32) were vaccinated with new preparations of the sameDNA vaccine they had received 1 to 2 years earlier. Thisbooster vaccination was administered to affirm that immuno-logical memory had been elicited by the initial vaccinationseries and to ensure robust responses to the DNA vaccineswith the intent to prove concepts rather than explore minimalrequirements for protection. Sera collected at the time ofboosting and 11 days later were evaluated for anamnestic an-tibody responses by ELISA, PRNT, and RIPA (Fig. 1). Beforethe DNA vaccine boost, all sera except CH93 had undetectablelevels of antibody as measured by VACV-infected-cell lysateELISA and PRNT (Fig. 1a). After the boost, all of the mon-keys in groups 1 and 2 had detectable levels of neutralizingantibodies and all except CH74 were positive by ELISA. Gene-specific RIPA indicated that the three monkeys vaccinatedwith the 4pox DNA vaccine had increased levels of antibody

TABLE 1. Rhesus macaques used in MPOV i.v. challenge: vaccination history and challenge outcome

Process IDa Sex/wt(kg)b Vaccinec

Vaccination (wk)Challenge

(PFU)

Challenge outcome

Initial series Boostd No. oflesionse Disease severity Day of death

postchallenge

Dosing expt CH42 F/5.8 Neg. cont. 0, 3, 6, 11 ND 5 � 108 0 Gravef 6CH03 F/4.5 Neg. cont. 0, 3, 6, 11 ND 5 � 108 0 Gravef 6CH85 F/4.8 Neg. cont. 0, 3, 6, 12 ND 5 � 106 198 Severe SurvivedCH64 F/5.5 Neg. cont. 0, 3, 6, 12 ND 5 � 106 125 Severe Survived

Vaccine evaluation L201-1 M/8.4 4pox DNA 0, 3, 6, 11 121 2 � 107 13 Mild SurvivedRC114 F/4.0 4pox DNA 0, 3, 6, 12 59 2 � 107 30 Moderate SurvivedCH93 F/4.1 4pox DNA 0, 3, 6, 12 59 2 � 107 1 Mild SurvivedCH63 F/4.0 L1R DNA 0, 3, 6, 12 59 2 � 107 100 Severe SurvivedCH74 F/4.5 L1R DNA 0, 3, 6, 12 59 2 � 107 212 Severe SurvivedCH28 F/5.3 Neg. cont. 0, 3, 6, 11 59 2 � 107 TNTC Graveg 7CH02 F/4.7 Neg. cont. 0, 3, 6, 11 59 2 � 107 TNTC Grave 10CH32 F/4.5 Neg. cont. 0, 3, 6, 11 ND 2 � 107 TNTC Grave 14CH39 F/4.1 Dryvax 0 ND 2 � 107 0 No disease SurvivedCH65 F/3.8 Dryvax 0 ND 2 � 107 0 No disease Survived

a ID, monkey identification number.b F, female; M, male.c Neg. cont., negative control DNA vaccine.d ND, not done.e TNTC, too numerous to count.f Hemorrhagic monkeypox.g Hemorrhagic monkeypox progressing to disseminated exanthem.

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FIG. 1. Memory antibody response to vaccination with poxvirus DNA vaccines. (a) Sera from rhesus macaques previously (1 to 2 years)vaccinated with the 4pox DNA vaccine, L1R DNA vaccine, negative control (Neg. cont.) DNA vaccine, or Dryvax collected immediately before(pre) and 11 days after (post) a booster vaccination were evaluated for anti-VACV antibodies by ELISA using VACV-infected-cell lysate antigenand VACV PRNT. ELISA end point and PRNT50 titers are shown. *, ELISA titer of �100 or PRNT titer of �20. na, not applicable because therewas no boost. (b) Pre- and postboost sera from three monkeys (L201-1, RC114, and CH93) vaccinated with the 4pox DNA vaccine were testedfor immunogen-specific antibodies by RIPA using lysates from COS cells transfected with plasmids expressing the four VACV genes that comprisethe 4pox DNA vaccine: pWRG/L1R, pWRG/A33R, pWRG/B5R, and pWRG/A27L. Lysate from COS cells transfected with empty-vector plasmid(pWRG7077) was used as a negative control antigen (�). (c) Pre- and postboost sera from two monkeys vaccinated with the L1R DNA vaccine


against each of the four immunogens (Fig. 1b). Sera from thetwo monkeys vaccinated with the L1R DNA vaccine had ele-vated levels of L1R-specific antibody after the boost (Fig. 1c).Thus, gene gun vaccination with the 4pox DNA vaccine or theL1R DNA vaccine elicited a memory response that was main-tained for at least a year and up to 2 years (i.e., monkeyL201-1).

Dosing experiment results. Before the challenge experi-ment, two dosing experiments were performed to (i) extend tothis MPOV isolate the previous reports (22, 24) that MPOVcauses severe monkeypox when administered by the intrave-nous (i.v.) route and (ii) determine the dose of MPOV-Z79sufficient to cause severe, but not overwhelming, disease. In thefirst experiment, two rhesus macaques were injected i.v. with ahigh dose (5 � 108 PFU) of MPOV-Z79. This dose causedorgan-hemorrhagic monkeypox, and the animals died on day 6before presenting generalized exanthema. The likely cause ofdeath was cardiovascular collapse secondary to multiple organfailure (e.g., liver) exacerbated by a bleeding diathesis andinability to recover from anesthesia. In the second experiment,two rhesus macaques were injected i.v. with 5 � 106 PFU. Arash erupted on day 6 and progressed to disseminated exan-thema with �100 lesions per animal. The animals survived withonly minor scarring. Clinical manifestations on the days fol-lowing the high- and low-dose challenges are shown in Fig. 2aand Table 1. By the World Health Organization scoring systemused during the smallpox eradication program, �25 lesionsrepresented mild disease, 25 to 99 lesions represented moder-ate disease, 100 to 250 lesions represented severe disease, and�250 lesions represented grave disease. Thus, 5 � 106 PFU issufficient to cause severe monkeypox and 5 � 108 PFU causesa hemorrhagic manifestation of the disease that is rapidly fataland does not resemble naturally occurring monkeypox orsmallpox.

Monkeypox challenge. Based on the dosing experiments(Fig. 2a), a dose of 2 � 107 PFU was chosen for the vaccineevaluation experiment. Vaccinated monkeys were challengedwith MPOV-Z79 by i.v. injection into the right or left saphe-nous vein. At 2-day intervals, whole-blood, serum, and throatswab samples were collected, and rectal temperature, pulse,and blood oxygen saturation were measured. Salient clinicaland laboratory findings are shown in Table 1 and Fig. 2b (alsosee Fig. 4). Monkeys vaccinated with the negative control DNAvaccine developed grave monkeypox and succumbed on days 7,10, and 14. Monkeys vaccinated with Dryvax showed no signsof clinical disease, indicating that VACV given more than ayear earlier could confer protective immunity against this ro-bust MPOV challenge dose. This confirms earlier findings thatvaccination of rhesus macaques with a commercial smallpoxvaccine confers protection against monkeypox after i.v. chal-lenge (22). Monkeys vaccinated with the L1R DNA vaccinedeveloped severe monkeypox and atypical lesions, but the an-imals recovered. This suggested that vaccination with L1R

alone can confer some protection against monkeypox. Themost important finding of this study was that monkeys vacci-nated with the 4pox DNA vaccine were protected not onlyfrom lethal monkeypox but also from severe disease (Fig. 2band Table 1). This is the first demonstration that vaccinationwith a combination of VACV immunogens, rather than thewhole infectious virus, is sufficient to protect nonhuman pri-mates against any poxvirus disease.

The cause of death for the negative control monkey (CH28)that died on day 7 was similar to that of the two monkeys(CH42 and CH03) that had received a high dose of virus(i.e., organ-hemorrhagic monkeypox). There was evidence ofhemorrhage in the lymph nodes, heart, lungs, urinary bladder,uterus, and digestive tract. In addition, there were hepatopa-thy, splenomegaly, lymphadenopathy, diffuse pulmonaryedema, and degeneration or necrosis of the bone marrow.Unlike the high-dose recipients, CH28 showed signs of pro-gression to severe exanthematous monkeypox. The monkeysthat died on days 10 and 14, CH02 and CH32, respectively,presented a disseminated exanthematous rash, marked lymph-adenopathy (up to 20 times normal size), mild splenomegaly,mild pulmonary edema, and a notable absence of remarkablepathology in other organs. Except for lymphadenopathy, whichis a clinical symptom of monkeypox (18), the necropsy findingsare similar to those of autopsies of human smallpox fatalitieswhere none of the vital organs appeared to be severely dam-aged and death was attributed to “toxemia” (10a).

The most dramatic clinical manifestation of monkeypox inthe challenged monkeys, other than death, was the generalizedvesiculopustular rash. The rash was first evident 6 days post-challenge and progressed from macules to papules, to vesicles,to pustules, and finally to crusts over 10 days. The crusts fell off,in some cases leaving scars. As in naturally occurring humanmonkeypox and smallpox, the distribution of lesions was pri-marily on the face (Fig. 2c) and hands (Fig. 2d) but rarely onthe abdomen. The sex skin, nipples, and buttocks were alsocommonly affected. In three monkeys (RC114, CH74, andCH63), the area of the leg surrounding the site of i.v. injectionwas a region with a relatively high number of lesions (Fig. 2b).For example, 52 of the 100 lesions on CH63 were distributedon the injected leg with fewer than five on the other leg (Fig.2e). We suspect that seeding of the tissues surrounding the siteof injection occurred at the time of injection, possibly by asmall amount of inoculum that missed the saphenous vein andentered blood or lymph capillaries. Although it became easierto count lesions as they progressed from macules to crusts,there was only one crop of lesions, which differentiates or-thopoxvirus-associated rash from other viral-infection-associ-ated rashes, such as chickenpox. The temporal distribution oflesions during the disease course is shown in Fig. 2b.

One notable difference between the monkeys vaccinatedwith the L1R DNA vaccine and all other monkeys in this studywas bleeding into the lesions beginning on day 14 (Fig. 2b and

(CH63 and CH74) were tested for L1R-specific antibodies by RIPA using lysates from COS cells transfected with a plasmid, pMPOX/L1Ro,expressing the MPOV L1R orthologous protein, L1Ro (13). Serum from a monkey (CH02) vaccinated with a negative control plasmid and serumfrom a monkey (CH63) vaccinated with pWRG/L1R collected after the initial series of vaccinations (9) were used as negative (�) and positive (�)controls, respectively. Lysate from COS cells transfected with the empty vector pWRG7077 served as a negative control antigen. Molecular massmarkers (M) are shown in kilodaltons on the left, and the positions of immunoprecipitated VACV proteins are shown on the right.


FIG. 2. Evolution of disease in rhesus macaques challenged i.v. with MPOV. (a) Monkeys vaccinated with negative control DNA vaccines wereinjected (day 0) with a high or low dose of MPOV-Z79. Graphic representations of rash distribution, lesion number, fever, elevated white bloodcell counts, and bleeding disorders over 3 weeks are shown. (b) Monkeys vaccinated with a negative control DNA vaccine, 4pox DNA vaccine, L1RDNA vaccine, or Dryvax were injected (day 0) with 2 � 107 PFU of MPOV-Z79. Throat swab and blood viremia data are included in panel b butnot in panel a. (c) Umbilicated lesions on face (monkey CH32; day 10 after challenge) 6 days after onset of rash. (d) Deep pustular lesions on CH74palm (day 10). (e) Disproportionate number of lesions on leg injected with virus (monkey CH63; day 14). (f) Bleeding lesions on CH74 palm (day14) 8 days after onset of rash.


f). Hemorrhage appeared to occur from below the pustules,and the lesions would flatten. This phenomenon was reminis-cent of reports of late-hemorrhagic-type smallpox in personswho had been successfully vaccinated with VACV (10). By day21, the lesions on the face, feet, and hands of CH63 and CH74were rapidly healing, and the monkeys recovered. This is incontrast to hemorrhagic smallpox in humans, which was almostalways fatal. Due to the small number of animals in this ex-periment, it is unclear if the hemorrhagic lesions are a normalvariation of late-stage monkeypox-associated rash in rhesusmacaques or somehow linked to preexisting L1R-specific im-munity.

Aside from the dramatic rash, other symptoms of disease inall three negative control animals included fever (�39.5°C) ondays 2 and/or 4 after challenge (Fig. 2b) and elevated whiteblood cell counts (Fig. 3a). Except for a trend toward low(�35%) hematocrit in most of the monkeys (Fig. 3b), therewere no consistent alterations in other parameters, includingweight, pulse, blood oxygen saturation, and platelet numbers,for any groups during the course of the study (data not shown).Analysis of blood chemistry indicated that one or more of thenegative control animals exhibited abnormal levels of analytesconsistent with the hepatopathy (Fig. 3c).

As shown in Table 1 and Fig. 2b and 3, the monkeys vacci-nated with the 4pox DNA vaccine had very mild or, in the caseof CH93, almost nonexistent clinical or laboratory indicationsof monkeypox. Note that the lesions that did develop in thegroup vaccinated with the 4pox DNA vaccine not only werefewer but also healed more rapidly than those in the low-dosegroup (Fig. 2a) or the L1R DNA vaccine group (Fig. 2b). Thetwo monkeys vaccinated with the L1R DNA vaccine wereclinically normal with the following exceptions: CH74 had 212lesions, a fever on days 2 and 8, and a sustained decrease inalbumin on days 12 to 16, and CH63 had 100 lesions, anintermittent fever from days 6 thru 28, and a transient decreasein albumin on day 16. The two monkeys vaccinated with Dry-vax did not present any signs of disease after challenge.

Virus shedding in protected and unprotected animals. Todetermine if the positive control vaccine, Dryvax, or any of thecandidate DNA vaccines prevented virus shedding, plaque as-says were performed on processed throat swab suspensions.The results are shown in Fig. 2 and 4a. No infectious viruseswere detected in the oral secretions of the two monkeys vac-cinated with Dryvax. Similarly, no infectious viruses were de-tected in monkey CH93, which was vaccinated with the 4poxDNA vaccine. The other two monkeys vaccinated with the4pox DNA vaccine and both monkeys vaccinated with the L1RDNA vaccine had infectious virus in oral secretions starting 4days after challenge. Interestingly, infectious viruses were notdetected in the monkeys vaccinated with a negative controlDNA vaccine until day 6, which was 2 days after virus was firstdetected in the DNA vaccine groups.

To determine if infectious virus was present in the blood ofchallenged monkeys, we performed plaque assays on wholeblood. No infectious viruses were detected in the blood frommonkeys that were vaccinated with the 4pox DNA vaccine,L1R DNA vaccine, or Dryvax (data not shown). In contrast,infectious viruses were detected in the blood of all three mon-keys vaccinated with the negative control DNA vaccine (Fig.4b).

Because virions could be present in the blood but not de-tected by plaque assay due to the presence of inhibitors (e.g.,neutralizing antibodies and/or complement), we assayed forthe presence of MPOV genomes in the blood using real-timePCR. Viral genomes were detected in the blood of all monkeysvaccinated with the negative control DNA vaccine. In the mon-key that developed hemorrhagic monkeypox (CH28), viral ge-nomes were detected as early as day 2 and continued to rise,apparently unchecked, until death on day 7. Viral genomeswere detected in two monkeys vaccinated with the 4pox DNAvaccine and both monkeys vaccinated with the L1R DNA vac-cine. In these four monkeys, levels of infectious virus in oralsecretions peaked before viral genomes were detected in theblood. The same three monkeys that had no infectious virusdetected in oral secretions (CH93 vaccinated with 4pox DNAvaccine and CH39 and CH65 vaccinated with Dryvax) also hadno MPOV genomes detected in their blood (Fig. 4c).

Monkeys vaccinated with DNA vaccines that survived chal-lenge but still shed virus (L201-1, RC114, CH63, and CH74)had detectable levels of infectious viruses in throat swabs atleast 2 days before viral genomes were detected in the blood(Fig. 4a and c). Moreover, these monkeys had infectious vi-ruses in their throats 2 days before infectious virus was de-tected in the throat swabs of the negative control monkeys.One possible explanation for the early presence of virus in thethroat swabs of partially protected animals is the detection ofinput challenge virions that remained infectious after beingopsonized by effector cells and cleared through the lungs. Inanimals that were better protected (CH93, CH65, and CH39),we speculate that the immune response was capable of inacti-vating the input virus by redundant mechanisms, which effec-tively eliminated all forms of infectious virus. In the negativecontrol monkeys, infectious virus was not detected in oral se-cretions until the time that the rash appeared, day 6, suggestingthat the virus could have been shed from lesions in the oral andpharyngeal mucous membranes.

Antibody responses before and after challenge. PRNT re-vealed that all of the monkeys except CH28 responded to theMPOV challenge by producing neutralizing antibodies (Fig.5a). CH28 developed rapidly progressing hemorrhagic mon-keypox and died on day 7, before detectable levels of antibod-ies were produced. Although the other two negative controlmonkeys produced antibodies capable of neutralizing �80% ofthe virus, the response was not detectable until 10 days afterchallenge, and the titers never exceeded 40. The rise in neu-tralizing antibody indicated that all of the monkeys in thisstudy, including those completely protected from clinical dis-ease, responded immunologically to the challenge virus.

To measure the immunogen-specific antibody responses, weprepared partially purified A27L, L1R, B5R, and A33R VACVantigens from E. coli and evaluated serum collected from thevaccinated monkeys by ELISA (Fig. 5b). Antibody responsesto all four immunogens were detected in only one of themonkeys vaccinated with the 4pox DNA vaccine (CH93). Thismonkey had only one lesion, no virus in oral secretions, and noviremia, making it the most completely protected monkeyother than the Dryvax-vaccinated monkeys. The other twomonkeys vaccinated with the 4pox DNA vaccine were positivefor A27L, B5R, and A33R but not L1R. Monkeys vaccinatedwith the L1R DNA vaccine had relatively high L1R-specific


antibody responses and were negative for the other three an-tigens. The two monkeys vaccinated with Dryvax exhibited verydifferent antigen-specific antibody responses. CH39 had levelsof antibodies undetectable by ELISA, and CH65 had relativelystrong antibody responses to all four antigens. These Dryvax-

vaccinated monkeys had VACV-infected-cell lysate ELISA ti-ters and PRNT titers within twofold of each other (Fig. 1a),indicating that the net VACV-specific antibody responses inthese monkeys were similar. The fact that CH39 showed noclinical signs of disease after challenge indicates that high

FIG. 3. Abnormal hematological findings in immunized and control monkeys challenged with MPOV. (a and b) Automated cell counts of wholeblood were determined; white blood cell (WBC) counts (a) and hematocrit (b) are shown. (c) Serum clinical chemistries. Monkeys were challengedon day 0 (bold vertical line). The dashed lines indicate normal high and low values for rhesus macaques. †, fatality.


levels of antibodies to A27L, L1R, B5R, and A33R were notrequired for protection.

Previous work had demonstrated that after the initial vacci-nation series, representative monkeys vaccinated with 4poxDNA vaccine or L1R DNA vaccine had antibody responsescapable of binding MPOV orthologous proteins and cross-neutralizing MPOV (13). To determine the levels of MPOV-neutralizing antibodies at the time of challenge, we performedMPOV PRNT with sera collected on the day of challenge (Fig.5c). Low levels of MPOV-neutralizing antibodies were de-

tected in all of the monkeys except those vaccinated with thenegative control DNA vaccines. Monkeys vaccinated with the4pox DNA vaccine had higher levels of MPOV-neutralizingantibodies than those vaccinated with the L1R DNA vaccineand were better protected. It is possible that the higher levelsof neutralizing antibodies in the monkeys vaccinated with the4pox DNA vaccine were responsible for the greater levels ofprotection in the 4pox DNA vaccine group compared to theL1R DNA vaccine group. Alternatively, the greater level ofprotection could be due to immune responses to one or moreof the other three immunogens in the 4pox DNA vaccine.

Summary. Remarkably little is known about the mechanismby which vaccination with VACV confers protection againstorthopoxviruses. Several lines of evidence, primarily naturalexperiments involving smallpox patients with underlying im-mune system defects, indicate that both the humoral and cell-mediated arms of the immune system play important roles inprotection (19). Recently, Belyakov et al. reported that anti-bodies were necessary for protection against disease in vacci-nated mice whereas CD4� and CD8� T cells were neithernecessary nor sufficient (1). In that same study, the CD4�- andCD8�-T-cell responses were important in conferring protec-tion against natural infection and were sufficient to protectagainst lethal disease in the absence of antibody (1). Here, wefocused on four VACV immunogens that are known to betargets of neutralizing or otherwise protective antibody re-sponses (7, 8, 11, 20, 25, 26, 30). We evaluated the humoral, butnot cell-mediated, responses following vaccination. In futurestudies, we will investigate whether the antibody responses aresufficient to protect or if cell-mediated responses elicited byone or more of the immunogens contained in the DNA vaccineare necessary to achieve the observed protection.

Vaccination with a single VACV gene that encodes a knowntarget of neutralizing antibodies, L1R, protected against lethal-ity but not against severe disease. The clinical symptoms of themonkeys vaccinated with the L1R DNA vaccine were similar tothose in the monkeys challenged with a fourfold-lower dose ofvirus (Fig. 2b). Thus, mitigation of disease could be due toreduction of the effective challenge dose by neutralization ofthe challenge virus. MPOV-neutralizing antibodies in the seraof monkeys vaccinated with L1R were detected at the time ofchallenge, but at very low levels. These sera had high levels ofanti-L1R antibodies as measured by immunogen-specificELISAs, indicating that the anti-L1R response elicited by theDNA vaccine might have contained a high proportion of non-neutralizing anti-L1R antibodies. These same sera had higherlevels of VACV-neutralizing antibodies (data not shown), in-dicating that to protect against monkeypox, it might be bene-ficial to vaccinate with the MPOV L1R ortholog. Similarly, theuse of L1R, A27L, A33R, and B5R orthologs homologous tothe challenge virus, whether it is VACV, MPOV, or variolavirus, might improve the efficacy of the vaccine.

We demonstrated here that a DNA vaccine comprised offour VACV genes and administered by gene gun is capable ofprotecting nonhuman primates against severe monkeypox.This is the first report demonstrating that a subunit vaccine iscapable of protecting nonhuman primates from any poxvirus-associated disease. The DNA-vaccinated monkey with thehighest prechallenge antibody responses, CH93, was almostcompletely protected (a single lesion, 1 day of fever, no virus

FIG. 4. Time courses of viremia in vaccinated and control monkeyschallenged with MPOV. (a) Infectious MPOV detected in throatswabs. (b) Infectious virus detected in whole blood by plaque assay. (c)MPOV genomes detected in whole blood by TaqMan PCR. Monkeyswere challenged with MPOV on day 0. †, fatality.


FIG. 5. Antibody responses before and after MPOV challenge. (a) Sera from monkeys vaccinated with 4pox DNA vaccine, L1R DNA vaccine,negative control (Neg. cont.) DNA vaccine, or Dryvax were collected 1 week before MPOV challenge, at the time of challenge (day 0), and at theindicated times after challenge. PRNT 80% neutralization titers are shown. (b) Immunogen-specific antibody titers in sera collected during theweek before challenge were determined by ELISA using partially purified A27L, L1R, B5R, or A33R protein expressed in E. coli. Each barrepresents the geometric mean titer obtained from at least two experiments. (c) MPOV-specific neutralizing antibody titers in sera collected at thetime of MPOV challenge (day 0). The bars represent the mean value of two to four determinations standard deviation.


detected in oral secretions, and no viremia). This finding leadsus to believe that a subunit (gene- or protein-based) poxvirusvaccine has the potential to mimic the protection afforded bylive VACV administered by scarification. Such a vaccine wouldcontribute greatly to vaccination strategies aimed at reducingthe health hazards of the present smallpox vaccine.

ACKNOWLEDGMENTS

We thank J. Geisbert for performing clinical chemistries, L. Hensleyand R. Fisher for help in blood sample processing, K. Stabler fortechnical assistance, M. S. Ibrahim for providing the optimized assayfor detecting the MPOV genome, and P. Rico for serving as attendingveterinarian. We also thank J. Huggins for providing us with MPOV-Z79, as well as helpful discussions regarding monkeypox disease innonhuman primates. The particle-mediated epidermal delivery device(gene gun) was kindly provided by Powderject Vaccines Inc., Madison,Wis.

Opinions, interpretations, conclusions, and recommendations arethose of the authors and are not necessarily endorsed by the U.S.Army.

The research described here was sponsored by the Military Biolog-ical Defense Research Program, U.S. Army Medical Research andMaterial Command, project no. 02-4-7I-095.

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smallpox dna vaccine protects nonhuman primates against lethal monkeypox

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